Research ArticleMicrobiological Quality and Risk Assessment forAflatoxins in Groundnuts and Roasted Cashew Nuts Meant forHuman Consumption

Modupeade Christianah Adetunji ,1,2 Ogechi Precious Alika,2 Ngozi Precious Awa,2

Olusegun Oladimeji Atanda,2 andMulundaMwanza1

1Department of Animal Health, School of Agriculture, Faculty of Agriculture, Science and Technology, North-West University,Mafikeng Campus, Private Bag X2046, Mmabatho 2735, South Africa2Department of Biological Sciences, McPherson University, Km 96, Lagos-Ibadan Expressway, Seriki Sotayo, PMB 2094,Abeokuta, Ogun State, Nigeria

Correspondence should be addressed to Modupeade Christianah Adetunji; ogunrinumodupe@gmail.com

Received 17 November 2017; Revised 23 February 2018; Accepted 1 April 2018; Published 3 June 2018

Academic Editor: Brad Upham

Copyright © 2018 Modupeade Christianah Adetunji et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Nuts are one of the commonly consumed snacks but poor handling and storage practices can make them prone to foodborneinfections. The study aimed at assessing the microbiological quality and risk assessment for aflatoxins in groundnuts and cashewnuts consumed in selected locations in Nigeria. The moisture content, colony counts, incidence of pathogenic bacteria, aflatoxincontamination, and risk assessment for aflatoxins were evaluated using standard methods. The moisture content and total viablecount ranged from 5.00–8.60% and 5.5–89 × 103 cfug−1, respectively, while the fungal count was between 4–24 × 103 and 1.0–4.5 ×102 cfug−1, respectively. Eleven fungal species belonging to 5 genera were isolated from the nuts, with Aspergillus flavus, Rhizopusoryzae, and Fusarium oxysporum having the highest percentage occurrence of 50%. In addition, the aflatoxin concentrationranged 0.1–6.8 and 29–33.78 ng kg−1 for cashew nuts and groundnuts, respectively. The margin of exposure (MOE) to aflatoxincontamination was 6.10 for groundnuts and 1000 for cashew nuts and the nuts consumers were at a risk of exposure to foodbornediseases and aflatoxin contamination withmean exposure values of 27.96 and 0.17 ng kg−1bwday−1, respectively.The risk of primaryliver cancer for groundnuts and cashew nuts consumers was also estimated to be 1.38 and 0.01 canceryear−1100,000−1person,respectively. This calls for mitigation measures from appropriate governmental organizations.

1. Introduction

Nuts are delicacies generally accepted by almost everyonedue to their high nutritive value and taste [1]. Apart fromdirect consumption, nuts are also useful for various industrialpurposes such as extraction of oils for home and industrialuses, cooking, soap production, and body cream [2]. Nuts arerich in proteins, fats, minerals, and low water content; hence,they are highly susceptible to microbial invasion especiallyfungal attack [1].

Nuts are susceptible to fungal attacks at different stagesand time.Theymay invade the nuts while still on the trees andthis usually occurs when the hard shells or pods of the nuts

are split open and the seeds attacked by insects or pests whichcreates room for the fungal spores to access the developingseeds. Other sources of contamination include harvesting,sorting, and washing of the nuts before storage. If the nutsare not properly handled at this stage, it could lead to mouldgrowth especially when seeds are not properly dried to therecommended moisture level before storage [2, 3]. Anothermajor source ofmould invasion could be during storagewhennuts are stored under conditions suitable for mould growthsuch as high temperature and humidity [4].

Both bacteria and fungi are responsible for microbialbiodeterioration of food crops leading to food and economiclosses, thereby reducing the export value of the food crops

HindawiJournal of ToxicologyVolume 2018, Article ID 1308748, 11 pageshttps://doi.org/10.1155/2018/1308748

2 Journal of Toxicology

[5]. The fungal species could penetrate the hard shell of thecashew nut to commence biodeterioration processes withoutnecessarily showing any form of mouldiness.

Mycotoxins are secondary metabolites produced by cer-tain filamentous fungi [6, 7]. Aflatoxins are considered oneof the main types of mycotoxins produced by fungi of thegenusAspergillus, especiallyAspergillus flavus andAspergillusparasiticus. Aflatoxins consumed by humans may lead todifferent forms of diseases like cancer, cirrhosis and otherliver diseases, spontaneous abortion, and immunosuppres-sion interference withmicronutrientmetabolism and stuntedgrowth [8].They have also been associated with diseases suchas aflatoxicosis in livestock, domestic animals, and humansthroughout the world. The adoption of proper measures forcollection, storage, transportation, and handling could helpreduce the risk of exposing nuts to attacks of mycotoxigenicfungi [9, 10]

Many countries have set maximum tolerable levels(MTLs) for mycotoxins of human health concern in foods,yet these regulations seemnot to confer the required safety onthe populace if large amount of foods with high susceptibilityto aflatoxins such as maize, groundnuts, and melons are con-sumed by the population [11]. AFB1 has been implicated asa potent liver carcinogen, causing hepatocellular carcinoma(HCC) in humans and a variety of animal species. It hasalso been classified as Group 1 human carcinogen by theInternational Agency for Research on Cancer (IARC) withconsequent negative effect such as adverse immune systemdisruption and stunted growth in children [12, 13]

Aflatoxins in foods are converted to aflatoxin-8,9-epoxidemetabolite in the liver which seems to be responsible formany of the toxic effects in the body [14]. Coexposure toaflatoxins and hepatitis B virus (HBV) is common in devel-oping countries and greatly increases HCC risk. Individualswith both exposures often havemultiplicatively greater risk ofdeveloping HCC than those exposed to aflatoxins alone [15–17].

Despite the availability of literatures on mycotoxin con-tamination of groundnuts from different parts of Nigeria[18–20] information on the aflatoxin contamination and riskassessment due to consumption of cashew nuts is scarce.However, Adebajo andDiyaolu [21] reported on the incidenceof aflatoxin and mould contamination of cashew nuts fromLagos State, Nigeria, but did not report on the risk ofexposures of the consumers. This work is therefore aimedat assessing the microbiological quality and risk assessmentfor aflatoxins in cashew nut and groundnut consumers inLagos and Ogun state, Nigeria. This will help to evaluate thecurrent safety level of consumers in the selected states andalso provide information to policymakers on appropriate lineof actions to embark on.

2. Sample Collection and Preparation

Nuts are among the most common snacks consumed byalmost everyone in Lagos State due to the busy nature of itsinhabitants. Lagos is a cross metropolitan state with diversekinds of migrants from within and outside the country whomigrated into the state to earn a living. As a result of the

busy schedule of the citizens, most people spendmore time atwork than at home and as a result eat less of homemade foodsand more of snacks such as biscuits, nuts, and fried foods.Since nuts are perceived to be healthier and also have pleasanttastes, people have a great tendency to consume them insteadof other snacks such as biscuits, chips, and popcorn. Due tothe high population density of Lagos, newmigrants now settlein Ogun state, a neighboring state to Lagos State, hence thechoice of part of the state in the sampling process.

Twenty-seven samples of roasted cashew nuts and 15samples of raw groundnuts were purchased from 9 and 5locations in Lagos State and Obafemi Owode Local Govern-ment area, Ogun state, Nigeria. Samples were collected atrandom from three different traders in each location. Thustriplicate samples of roasted cashew nuts were purchasedfrom the 5 zones that make up Lagos State, namely, “Eti-Osa”(“Ikota,” “Obalende,” and “Ajah”), “Badagry” (Market and“Topo”), “Ibeju-Lekki” (“Awoyaya and Lakowe”), “Mushin,”and “Ikeja,” while groundnut samples were purchased fromthe five major markets (“Ajebo,” “Fidiwo,” “Obafe,” “Ogun-makin,” and “Mowe”) in “Obafemi Owode” Local Govern-ment, Ogun State. Samples collected from the three differenttraders in each location were mixed together to form acomposite sample/location. The composited samples wereblended aseptically using Marlex Electronic Mixer Grinder(China) and 50 g portions kept in Ziplock envelopes. Samplesthat were not used immediately were kept at −20∘C prior toanalysis.

2.1. Moisture Content. Moisture content was determined outaccording to AOAC (2000) method. Briefly, samples (5 g)were oven-dried in a preweighed dish at 105∘C for 4 h andcooled in a desiccator continuously until a constant weightwas obtained. The moisture content was calculated fromreduction in weight and expressed as a percentage of theoriginal weight as illustrated below:

% Moisture Content =𝑊2 −𝑊3𝑊2 −𝑊1

× 100, (1)

where

𝑊1 is weight of empty Petri dish;𝑊2 is weight of sample + Petri dish before drying;𝑊3 is weight of sample + Petri dish after drying.

2.2. Determination of Total Viable and Fungal Counts. Thetotal viable count (TVC) and fungal counts were determinedby the dilution plate technique [22]. One gram of eachblended nut was added to 9ml of sterile water in a test tubeand the solution was decimally diluted. Twenty millilitresof sterilized molten nutrient agar and Potato Dextrose Agar(PDA) plates supplemented with 0.01% chloramphenicolwere cooled to 45∘C and poured separately unto platescontaining 1ml aliquots of each sample in triplicate and theplates gently swirled and allowed to solidify.The nutrient agarplates were incubated at 30∘C for 48 h for determination ofTVC and Potato Dextrose agar plates at 28∘C for 48 h fordetermination of fungal counts.

Journal of Toxicology 3

The total viable and fungal counts were determined usingthe following formula:

cfu/g

=Number of colonies × reciprocal of the diluting factor

Plating volume (1ml).

(2)

2.3. Isolation of Fungi by Direct Plating Method. This wasdetermined using the method described by Adebajo andDiyaolu [21]. Seven whole nuts were obtained randomly forthe assay of each nut. The cotyledons (2 per nuts) werehand separated, and the surfaces were disinfected with anaqueous solution of sodium hypochlorite 2% for 60 s. Thiswas followed by three rinses in sterile distilled water, beforefour cotyledons were plated equispaced on the medium(PDA agar plus chloramphenicol 0.01%). The Aspergillus andPenicillium cultures on PDA were incubated unilluminatedat 30 and 25∘C, respectively, for 7 days.The observed colonieswere purified on PDA agar plates to obtain pure cultures.

Identification of the fungal isolates was based on theexamination of both the macroscopic characteristics (colonycolour, morphology, and size) and microscopic characteris-tics (conidia morphology and size) features of the isolates onspecific media and comparison with appropriate identifica-tion keys [18, 23, 24].

Isolates of Aspergilli (mainly black Aspergillus) on PDAwere identified as A. niger clade [25]. The Fusarium andPenicillium isolates on PDA plates were also identified togenus level using appropriate manuals and references [22, 26,27].

2.4. Determination of Aflatoxigenic Potential of Cashew NutIsolates. Isolates belonging to theAspergilli section Flavi wereexamined for aflatoxigenicity on Neutral Red DesiccatedCoconut Agar (NRDCA), after 3 days of incubation bychecking for fluorescence of agar under long wavelength UVlight (365 nm).

The data obtained from toxigenic screening of A. flavusisolates on NRDCA was used to determine the ratio ofaflatoxigenic isolates to nonaflatoxigenic isolates in the nutsamples.

2.5. Determination of Percentage Fungal Occurrence. Thiswas determined as described by Atanda et al. [28]. Thepercentage of fungal occurrence was calculated by dividingthe occurrence of individual isolate per sample with the totalnumber of all fungal isolates in the sample which was thenexpressed as a percentage using the following formula:

𝑋

𝑌× 100, (3)

where

𝑋 is total number of individual isolate per sample;𝑌 is total number of all fungal isolates per sample.

2.6. Test for Pathogenic Bacteria in Cashew Nuts. Aliquots(0.1ml) of decimally diluted samples were spread plated

on Mannitol salt agar at 32∘C for 72 h for presumptivestaphylococcal count. Colonies surrounded by bright yellowzones were counted. Confirmation of Staphylococcus aureuswas a positive coagulase test [29].

The presumptive coliforms were counted on MacConkeyagar No. 3 at 37∘C for 24 h [29]. Confirmation of Escherichiacoli was by fermentation of lactose and indole at 44∘C after24 h incubation.

2.7. Aflatoxin Determination by ELISA Method. Suspectedaflatoxigenic fungi from both nuts were selected for aflatoxinanalysis using the Enzyme Linked Immounosorbent Assay(ELISA) technique. Commercially available kit Agraquant�,total aflatoxin test kit (4–40 ng/kg), was used for the quanti-tative analysis of aflatoxins.

2.7.1. Sample Preparation/Extraction. Five grams of theground sample was weighed into a tightly sealed jar towhich 25ml methanol (70%) was added and allowed to settlefor 5–10min. The sample was then filtered using a No. 1Whatman filter paper. Each of the fifty microlitres of thefiltrate and aflatoxin standards in separate dilution wells wasmixed with 100 𝜇l of the conjugate. Hundred microlitres wastaken from the filtrate/standard-conjugatemixture and put inthe antibody coated wells and incubated at room temperature(30∘C) for 15min. The contents of the wells were discardedand the wells washed with distilled water 3-4 times. Hundredmicrolitres of the substrate was added to each well andincubated for 5min to allow for colour change (differentshades of blue to colourless) and hundred microlitres of thestop solution was further added to each well to stop thereaction and this converted the blue end point to yellow andthe wells were transferred to the ELISA reader machine toquantify the amount of toxins in the samples

2.7.2. Quantification of Aflatoxins. This result was read at450 nm, using ELISA plate reader. Optical densities of stan-dards (0 ngg−1, 4 ngg−1, 10 ngg−1, 20 ngg−1, and 40 ngg−1)and those of samples were also recorded. A graph of thestandard concentration versus optical densities was plottedand extrapolations were made to determine levels of totalaflatoxins in the roasted cashew nuts samples.

2.8. Exposure Assessment of Nut Consumers to AflatoxinContamination. The exposure levels of the nut consumerswere determined as described by by Adetunji et al. [30].The quantity of aflatoxins in the groundnut samples wasmultiplied by the average consumption rate of the nuts inNigeria (groundnuts, 52 g/person/day) according to WHO[31] which was then divided by the average body weightof 60 kg for adults [30]. There is paucity of information inliterature on the average daily consumption rate of cashewnuts in Nigeria; hence the average consumption rate forcashew nuts was extrapolated by comparing the ratio ofannual production of the two nuts.The annual production ofgroundnuts was reported as 3,028,571 tons (http://www.fact-fish.com/statisticcountry/nigeria/peanuts%2C%20production%20quantity) while that of cashew nuts was 120,000 tons

4 Journal of Toxicology

(http://agriculturenigeria.com/farming-production/crop-production/cash-crops/cashew), thus with a production dif-ferential ratio of approximately 1 : 25. Thus, the consumptionrate for cashew nuts in this report was estimated as 2.08 gperson−1day−1, obtained by dividing the average daily adultconsumption rate for groundnuts with the productiondifferential ratio of the two nuts. The exposure assessment(PDI) was thus calculated as illustrated below:

PDI𝑚 =𝐶𝑚 × 𝐶𝑐

Bw, (4)

where

PDI𝑚 is probable daily intake for aflatoxins(ngkg−1bwday−1);

𝐶𝑚 is mean level of aflatoxins in the nuts per location(ng g−1);

𝐶𝑐 is average consumption of nuts in Nigeria (gday−1);

Bw is body weight for an adult (kg).

2.9. Risk Characterization for Aflatoxins. Risk characteriza-tion for genotoxic and carcinogenic compounds such asaflatoxins is based on themargin of exposures (MOEs), whichwas calculated by dividing the Benchmark dose lower limit

(BMDL) for aflatoxins- 170 ngkg−1bwday−1 [32] by the toxinexposure.

In cases where MOEs were lower than 10,000, a publichealth concern is indicated which implied that aflatoxinexposures above 0.017 ngkg−1bwday−1 (as obtained by divid-ing 170 ngkg−1bwday−1 by 10,000) represented a risk of publichealth concern [33].

2.10. Estimated Liver Cancer Risk due to Consumption of Nuts.The estimated liver cancer risk for Nigerian adult consumerswas calculated for aflatoxins because the ingestion of the toxincan be traced to the development of liver cancer [32, 34].Thisinvolved estimating the population cancer risk per 100,000which was obtained by multiplying the PDI𝑚 value withthe average hepatocellular carcinoma (HCC) potency figurefrom individual potencies of HBsAg-positive and forHBsAg-negative groups.

The JECFA estimated potency values for AFB1 whichcorresponded to 0.3 cancersyear−1100,000−1population/ngkg−1bwday−1 (uncertainty range: 0.05–0.5) in HBsAg-positive individuals and 0.01 cancersyear−1100,000−1popula-tion/ngkg−1bwday−1 (uncertainty range: 0.002–0.03) inHBsAg-negative individuals [32, 34] were adopted for thiscalculation. Also, the HBsAg+ prevalence rate of 13.6% forNigeria [35] was adopted and 86.4% (100 − 13.6%) wasextrapolated for HBsAg-negative groups. Hence the averagepotency for cancer in Nigeria was estimated as follows:

Average potency = (0.3 × 0.136) + (0.01 × 0.864)

= 0.04944 cancers per year per 100,000 population per ng AFB1 kg−1bwday−1.

(5)

Thus the population risk was calculated using the followingformula:

Population risk = Exposure × Average potency. (6)

2.11. Statistical Analyses. The data obtained from the analysiswere reported as means of three replicates and subjected toanalysis of variance (ANOVA) at 95% confidence level to testfor significance using SPSS 16.0 software (Windows version,SPSS, IL, USA). Colony counts of nuts were calculated ascfu/g.The relationship between aflatoxigenicity ofAspergillusisolates and aflatoxin concentrations in cashew nuts wasevaluated using Pearson correlation analysis.

3. Results

3.1. Microbiological Quality of Cashew Nut

3.1.1. Moisture Content and Total Viable Count. The averagemoisture content of the roasted cashew nuts from LagosState ranged 5.0–8.6% and were not significantly different(𝑃 ≥ 0.05) from each other. The mean moisture content ofcashew nuts was 6.04% with “Ajah” (5.0%) and “Obalende”(8.6%), having the lowest and highest moisture content. The

groundnut samples fromOgun state had an average moisturecontent of 6.12%.

Roasted cashew nuts from “Ikeja” had the highest TVCcount (64.5 × 103 cfug−1) while “Obalende” had the lowestcount of 5.5 × 103. Furthermore, a mean TVC count of 29.67× 103 cfug−1 was recorded for cashew nuts from Lagos Statewhile the mean viable bacterial count for the groundnuts was71.20 × 103 cfug−1 with “Mowe,” having the highest viablecount of 89.00 × 103 cfug−1. The fungal counts also ranged1.0 × 102 cfug−1–4.5 × 102 cfug−1 with a mean count of 2.30× 102 cfug−1 for cashew nuts while groundnuts had countsthat ranged from 4.00 × 103 cfug−1 for “Fidiwo” to 24.00 ×103 cfug−1 for “Mowe” (Table 1).

3.1.2. Incidence of Pathogenic Bacteria in Roasted CashewNuts. The incidence of pathogenic bacteria in the roastedcashew nuts is shown in Table 2. Coliforms were present inmost samples in Lagos State except for those collected from“Ajah,” while Staphylococcus aureuswas found in cashewnutsfrom only 2 locations (“Awoyaya” and “Badagry”) out of the9 sampled locations (22.22%) in the state. “Topo” had thehighest coliform count of 12 × 102 cfug−1 while “Obalende”and “Badagry” had the lowest counts of 1 × 102 cfug−1.

Journal of Toxicology 5

Table 1: Moisture content and colony counts of groundnuts and cashew nuts.

Groundnut Cashew nut

Location Moisturecontent

Total viableCount

(×103 Cfu/g)

Fungal count(×103 Cfu/g) Location

MoistureContent(%)

Total viablecount

(×103 cfu/g)

Fungal count(×102 cfu/g)

“Ajebo” 5.10a 70.00a 5.00a “Mushin” 5.2a 37.5d 3.0a

“Fidiwo” 6.12a 58.00a 4.00a “Ajah” 5.0a 25.0c 4.5b

“Obafe” 5.63a 78.00b 24.00c “Ikota” 5.3a 9.5b 1.0a

“Ogunmakin” 6.57a 61.00a 4.00a “Lakowe” 6.1a 13.5b 1.5a

“Mowe” 7.2a 89.00b 10.00b “Awoyaya” 6.3a 49.5e 4.0b

“Badagry” 5.6a 52.5e 1.5a

“Topo” 5.8a 9.5b 1.2a

“Obalende” 8.6b 5.5a 1.5a

“Ikeja” 6.5a 64.5f 2.5a

Mean 6.12 71.2 9.40 Mean 6.04 29.67 2.30Mean values with the same superscript along the same column are not significantly different (𝑃 < 0.05).

Table 2: Incidence of pathogenic bacteria in roasted cashew nuts from Lagos State, Nigeria.

Market Staphyloncus aureus count (×102 cfu/g) Coliform count (×102 cfu/g)“Lakowe” 0 4a

“Topo” 0 12b

“Obalende” 0 1a

“Ikota” 0 10b

“Awoyaya” 2a 3a

“Badagry” 1a 1a

“Ajah” 0 -“Mushin” 0 2a

“Ikeja” 0 2a

Mean values with the different superscripts along the same column are significantly different (𝑃 ≥ 0.05).

3.1.3. Fungal Occurrence. Nine fungal species belonging to 5major genera were isolated from the roasted cashew nuts inLagos State while 7 were isolated from the groundnut samples(Table 3). The common organisms to both nuts were asfollows: Aspergillus carbonarius, Aspergillus niger, Aspergillusflavus, Penicillium sp., and Curvularia lunata. Fusarium spp.was not found in the groundnut samples. The dominantfungi found in Lagos State were as follows: Aspergillusflavus, Rhizopus oryzae, and Fusarium oxysporum with 50%occurrence rates. “Lakowe” had the highest occurrence offungal species (55.55%) while samples from “Badagry” and“Ajah” had the least occurrence (9%).The dominant fungi inthe groundnut samples were A. flavus and A. niger with 60%occurrence each.

3.2. Aflatoxin Producing Potential of CashewNut Isolates. Theaflatoxigenicity test carried out on the Aspergillus isolatesfrom the cashew nuts showed that only A. flavus species(55.55%) had the potential to produce aflatoxins (Table 4).The positive isolates were stored in autoclaved glycerol water(10% glycerol) in an Eppendorf tube and stored at −80∘C forsubsequent use.

3.3. Aflatoxins Concentration of Nuts. The aflatoxin concen-tration of the cashew nuts ranged from 0.1 ngg−1 to 6.8 ngg−1with “Ikeja” and “Lakowe” having the least (0.1 ngg−1) andhighest (6.8 ngg−1) concentrations while the aflatoxin con-centration of the groundnuts ranged 29–33.78 ngg−1 (Table 5)with “Ajebo” having the least (29 ngg−1) and “Mowe”(33.78 ngg−1) the highest aflatoxins concentration.

3.4. Correlation between Aflatoxigenic Potential of FungalIsolates and Aflatoxin Concentration of Cashew Nuts. Asignificant (𝑃 < 0.01) inverse correlation (𝑟 = −0.980) existedbetween aflatoxin producing potential ofAspergillus flavus onNRDCA and the aflatoxin concentration of cashew nuts fromthe different locations (Table 6).

3.5. Exposure Assessment of Nuts Consumers to Aflatoxins.The range of exposures of consumers to aflatoxin con-tamination due to consumption of groundnuts in ObafemiOwode Local Government, Ogun State, and cashew nuts inLagos State ranged 25.13–29.28 ngkg−1bwday−1 (Table 7) and0.00–0.24 ngkg−1bwday−1(Table 8) with a mean of 27.96 and

6 Journal of Toxicology

Table3:Percentage

occurrence

offung

aliso

latesinNigeriannu

ts.

Sampletype

Locatio

n

Fung

alIsolate(%)

A.flavus

A.niger

A.Pa

rasiticu

sA.

fumiga

tus

Penicilliu

mspp.

A.cand

idus

A.car-

bona

rius

Curvularia

lunata

Rhizopus

oryzae

Fusariu

moxysporum

Fusariu

mverticil-

lioides

Cashew

nut

“Mushin”

ND

33.3

aND

ND

ND

ND

ND

ND

ND

∗66.7

bND

“Top

o”ND

ND

ND

ND

ND

ND

25a

ND

50b

ND

25a

“Awoyaya”

25a

ND

ND

25a

25a

ND

ND

ND

ND

ND

25a

“Lakow

e”16.7

aND

ND

33.3

bND

ND

16.7

aND

16.7

a16.7

aND

“Obalend

e”25

a25

aND

ND

ND

ND

ND

25a

25a

ND

ND

“Badagry”

ND

100

ND

ND

ND

ND

ND

ND

ND

ND

ND

“Ikota”

25a

25a

ND

ND

ND

ND

ND

ND

25a

25a

ND

“Ajah”

ND

ND

ND

ND

ND

ND

ND

ND

ND

100a

ND

∗∗Overall

occurrence

(%)

5037.5

ND

12.5

12.5

ND

2512.5

5050

25

Groun

dnut

“Ajebo

”28.57

42.85

ND

ND

ND

ND

14.28

14.28

ND

ND

ND

“Fidiwo”

83.33

ND

16.67

ND

ND

ND

ND

ND

ND

ND

ND

“Obafe”

4.76

4.76

ND

ND

80.95

ND

ND

9.52

ND

ND

ND

“Ogunm

akin”

ND

100.00

ND

ND

ND

ND

ND

ND

ND

ND

ND

“Mow

e”ND

ND

ND

ND

87.50

12.5

ND

ND

ND

ND

ND

Overall

occurrence

(%)

6060

20ND

4020

2040

ND

ND

ND

ND:not

detected;m

eanoccurrence

with

sames

uperscrip

talong

thes

amer

owisno

tsignificantly

different

(𝑃≥0.05).∗Frequencyof

occurrence

=(to

talnum

bero

feachorganism

inthes

ample)/(totalnum

bero

fallorganism

sinthes

ample).∗∗Overalloccurrence

=(no.of

locatio

nsthateach

organism

isfoun

d/(totaln

o.of

locatio

nssampled).

Journal of Toxicology 7

Table 4: Aflatoxigenicity of fungal isolates from cashew nuts fromLagos State on Neutral Red Desiccated Coconut Agar (NRDCA).

Location Aflatoxin producing potential“Ikeja” Negative“Obalende” Positive“Mushin” Negative“Badagry” Market Positive“Ikota” Positive“Mushin” Positive“Lakowe” Positive

Table 5: Aflatoxin concentration of nuts.

Cashew nut Groundnut

LocationAflatoxin

concentration(ng/g)

LocationAflatoxin

concentration(ng/g)

“Lakowe” 6.80b “Ajebo” 29.00a

“Obalende” 5.80b “Fidiwo” 33.00a

“Ikota” 5.40b “Obafe” 32.64a

“Ikeja” 0.1a “Ogunmakin” 32.88a

“Badagry” 6.4b “Mowe” 33.78a

Mean concentrations with same superscript along the same column are notsignificantly different (𝑝 ≥ 0.05).

Table 6: Correlations analysis between aflatoxins concentration andaflatoxigenic potential of isolate.

Aflatoxinconcentration

Aflatoxinpotential

Aflatoxinconcentration

Pearson correlation 1 −.980∗∗

Sig. (1-tailed) .002𝑁 5 5

Aflatoxin potentialPearson correlation −.980∗∗ 1Sig. (1-tailed) .002𝑁 5 5

∗∗Correlation is significant at the 0.01 level (1-tailed). 𝑟 = −.980,𝑁 = 5, 𝑃 <0.01.

0.17 ngkg−1bwday−1, respectively, which were higher thanthe permissible exposure level of 0.017 ngkg−1bwday−1. Themargin of exposures of the groundnut consumers (6.10) ascompared with MOE of cashew nut consumers (1000) wasmuch lower than the permissible limit of 10,000 (Tables 7 and8).

Furthermore, the HCC prevalence rate for ground-nut consumers in Obafemi Owode Local Government,Ogun State, ranged 1.24–1.45 cases 100,000−1peopleyear−1with a mean value of 1.38 cases 100,000−1peopleyear−1while there is little or no risk of HCC (0.00–0.01 cases100,000−1peopleyear−1) for the cashew nut consumers inLagos State.

4. Discussion

The moisture contents of the roasted cashew nuts wereabove the permissible recommended moisture limit of 5%[36]. Similarly the higher moisture content of the groundnutsamples (6.12%) could be as a result of its raw nature as it hasnot yet undergone any form of processing. A similar range ofmoisture content (6.48–7.05%) was reported for groundnutsfrom different agroecological zones of Nigeria by Oyedele etal. [18]. However, the moisture content of our roasted cashewnuts was higher than the findings of Adebajo and Diyaolu[21] who reported a range of moisture content of 4.1–6.8%with a mean of 5.4% in cashew nuts from the same state.Oluwafemi et al. [5] also reported a lower moisture contentrange of 3.7–4.3 and 3.2–5.4% for cashew nuts from Ogunstate during the dry and raining seasons.

Thehighmoisture contents of the samplesmay also be as aresult of improper packaging or lack of use of good packagingmaterials. Nuts are colloids materials (hygroscopic), whichtend to absorb moisture from the surrounding atmosphereuntil it reaches equilibrium, Oladapo et al. [37].Themoisturelevels of the nuts could also be influenced by the harvestingmethods of the farmers.

The difference in the microbial load of some of thesamples may be due to the varying climatic conditions inthe different zones across the two states. “Ikota,” “Ajah,” and“Obalende” (“Eti-Osa” zone) are situated in the humid forestzones of Lagos State.The high humidity in this zone may be acontributing factor to the highermicrobial counts of this zoneand probably coupled with improper drying, poor handling,poor processing techniques, and storage methods of the nuts.Several authors had reported a dense population of fungi ingroundnut samples [2, 18, 38] which also corroborated thefindings of this research work. The bacteria and fungi loadof the cashew nuts were however less than the 103 cfu/g and105 cfu/g limits recommended in foods by the InternationalCommission on Microbiological Specification for Foods(ICMSF, 1998). The lower counts of viable organisms in thecashew nuts may be due to the presence of anacardic acidsand phenolic compounds present in cashewnut oils which areinhibitory action against Gram-positive bacteria, yeast, andfungi. Anacardic acids with distinct lateral chains have beenshown to be effective against the growth of Staphylococcusaureus, Propionibacterium spp., Streptococcus mutans, andBrevibacterium ammoniagenes [39]. Although the microbialcounts of the nuts were lower than the recommended limitat the time of purchase and analysis, there is the possibilityof probable increase in the microbial load of the remain-ing market samples due to poor handling practices of theproducts by the sellers. A larger percentage of the sampleswere purchased from hawkers who vended the products inpolyethylene plastics in the sun. Continual exposure of thenuts to the sun during the day and recondensation in theevenings could support increase in the microbial load ofthe samples. With improper management of storage tem-peratures and humidity, aflatoxins producing moulds mayrapidly reproduce representing a food safety problem anda major risk for the production of aflatoxins. Furthermore,Aspergillus spp. can easily grow at optimal conditions of

8 Journal of Toxicology

Table 7: Risk Assessment due to consumption of groundnuts from Obafemi Owode Local Government Area, Ogun State, Nigeria.

LocationMean aflatoxinconcentration

(ngg−1)

1PDI(ngkg−1bwday−1)

2Margin ofexposure(MOE)

3Population risk forprimary liver

Cancer(canceryear−1100,000−1

population)“Ajebo” 29.00a 25.13 6.76 1.24“Fidiwo” 33.00a 28.60 5.94 1.41“Obafe” 32.64a 28.29 6.01 1.40“Ogunmakin” 32.88a 28.50 5.97 1.41“Mowe” 33.78a 29.28 5.81 1.45Mean4 32.26 27.96 6.10 1.38Values with different superscripts along the same column are significantly different at 𝑃 < 0.05. 1Probable Daily Intake (PDI) calculated by multiplying theaflatoxin concentration in groundnuts by consumption rates of groundnuts in Nigeria (52 g/person/day). 2Margin of exposure (MOE) calculated by dividingthe benchmark dose lower limit (BMDL) for aflatoxins by AFB1 exposure. 3Population risk for primary liver cancer = exposure × average potency (0.04944).4Mean of aflatoxin concentration.

Table 8: Risk Assessment due to consumption of cashew nuts from Lagos State, Nigeria.

Location Mean aflatoxin(ngg−1)

1PDI(ngkg−1bwday−1)

2Margin ofexposure(MOE)

3Population risk forprimary liver cancer

(canceryear−1100,000−1population)

“Lakowe” 6.80b 0.24 708.33 0.01“Obalende” 5.80b 0.20 850.00 0.01“Ikota” 5.40b 0.19 894.73 0.01“Ikeja” 0.1a 0.00 0.00 0.00“Badagry” 6.4b 0.22 772.72 0.014Mean 4.9 0.17 1000 0.01Values with different superscripts along the same column are significantly different at 𝑃 < 0.05. 1Probable Daily Intake (PDI) calculated by multiplying theaflatoxin concentration in cashew nuts by consumption rates of cashew nuts in Nigeria (2.08 g/person/day) as estimated byWHO 2008 and divided by assumedbody weight for Nigerian adults (60 kg). 2Margin of exposure (MOE) calculated by dividing the benchmark dose lower limit (BMDL) for aflatoxins by AFB1exposure. 3Population risk for primary liver cancer = exposure × average potency (0.04944). 4Mean of aflatoxin concentration.

27–33∘C, pH range of 5-6 andwater activity of 0.82–0.99 [40].Contamination of nuts by moulds may also occur early inthe field and deterioration could develop during prolongedstorage leading to the production of mycotoxins [41].

The high incidence of A. flavus, A. niger, Penicillium spp.,and Rhizopus oryzae and scant occurrence of Fusarium spp.reported in our nuts complemented the report of previousauthors [2, 38], who reported a similar trend of fungaloccurrence; however, Oyedele et al. [18] reported a higherincidence of Fusarium spp. (45%) in groundnut samplesacross the agroecological zones of Nigeria.

Although cashew nut is known to be an excellent sub-strate for microbial growth, outbreak of serious infectionsthrough its consumption had rarely been recorded. However,the probable presence of E. coli in the samples from LagosState is a source of concern as it is transmitted to humans pri-marily through consumption of contaminated foods. Symp-toms of the diseases caused by this Shiga toxin-producingE. coli include abdominal cramps and diarrhoea that mayin some cases progress to bloody diarrhoea (haemorrhagiccolitis). Fever and vomiting may also occur.

Staphylococcal food poisoning is also one of the mostcommon causes of reported foodborne diseases. Contami-nated equipment and environmental surfaces also can leadto S. aureus infections whose symptoms include diarrhoea,vomiting, nausea, and abdominal cramping. Freire andOfford [41] and Oluwafemi et al. [5] had earlier reportedon higher counts of S. aureus in Brazilian (5.3 × 103–1.2 ×104 cfug−1) and Nigerian (1–14 × 104 cfug−1) roasted cashewnuts, respectively. Processing conditions such as roasting andsalting could be responsible for the low count of S. aureus inour samples in addition to the presence of anacardic acid.Staphylococcus aureus is the most osmotolerant foodbornepathogen and the outbreaks of staphylococcal food poisoningare often linked to foods of reduced water activities [5, 42]and this could lead to septicaemia in humans. It is ideal thatnut vendors inculcate simple sanitary habits such as properdisposal of fecal waste and constant washing of hands withclean water and soap especially after visiting the toilet or incontact with any fecal objects. Food processors and handlersare also advised to stay away fromprocessing foodswhen theyhave cold, flu, cough, and diarrhoea to avoid contaminationof foods with the causative organisms.

Journal of Toxicology 9

This report further reemphasizes the suitability ofNRDCA for screening of toxigenic fung in foods beforefurther analyses with HPLC or other sophisticated methods.It is also readily more available as it can be easily compositedas described by Atanda et al. [28]

Eighty percent (80%) of the tested samples had aflatoxinconcentrations above the European Union (EU) permissiblelimit of 4𝜇g/kg, while 100% of the groundnut sampleshad aflatoxin concentration higher than the recommended20𝜇gkg−1 permissible limit set by the Standard Organiza-tion of Nigeria for unprocessed raw groundnuts. Similarly,higher concentration of aflatoxins (11.33–20.67𝜇gkg−1) wasreported for cashew nuts from Venezuela [43] while a lowerconcentration ofAFB1 (0.26–0.32 𝜇gkg

−1) and total aflatoxins(0.5–0.84 𝜇gkg−1) was reported for imported cashew nutsfrom Istanbul, Turkey [44]. A higher aflatoxin concentra-tion was however reported for groundnut samples fromKenya (Max; 250 𝜇gkg−1), Iraq (10.3 𝜇gkg−1), and Nigeria(216.1 𝜇gkg−1) [2, 18, 38]. Aflatoxins are highly toxic, fungalmetabolites commonly known to adversely affect the healthand nutrition status of humans and other animals in variousways. High concentration of aflatoxins is most often foundin plants with very nutritive seeds such as maize, nuts, andcereal grains in Africa [45]. Symptoms of aflatoxicosis aregastrointestinal infections including vomiting and abdominalpain. The toxin had also been incriminated as a potentialmutagenic, carcinogenic, and teratogenic threat to humans[46].

The high concentration of aflatoxins in the nuts especiallygroundnuts is reflected in the high PDI and MOE values ofthe nuts. Groundnut consumers in the local government stateare therefore at high risk of exposures to aflatoxicosis and livercancer and other forms of disabilities due to consumptionof the contaminated nuts. The evaluation of risk assessmentsof aflatoxins by international bodies has been based on thecarcinogenic potencies developed by JECFA, but, recently, theEC Scientific Panel on Contaminants in the Food Chain [32]developed a benchmark dose (BMD) approach. The BMD,which is a mathematical model originally put forward byCrump [47], represents an estimate of the dose requiredto produce a small response (1–10%) above the control.The BMD lower limit of 170 ngkg−1bwday−1 refers to thecorresponding lower limits of a one-sided 95% confidenceinterval on the BMD. For the evaluation of human andexperimental animal data, the EFSA Scientific Committeethus proposed the use of the BMD methodology to derive areference point on the dose-response curve. In addition, theBMD approach is recommended in the US EPA’s ProposedGuidelines for Carcinogen Risk Assessment [48] regardingmodelling tumour data and other (noncancer) responsesthought to be important precursor events in the carcinogenicprocess.

Although the risk of exposure through cashew nut con-sumption was low in Lagos State, there is still cause for con-cern because the smallestmolecule of the aflatoxinmetabolitecould evoke changes in human DNA leading to mutations,selective cellular proliferation, and cancer [49]. Aflatoxin B1has a reactive metabolite that interacts directly with DNA;

hence, it is assumed that there is no safe dose above zero.The recommendation of JECFA with regard to safe levelof aflatoxins in foods is to reduce “As Low As ReasonablyAchievable (ALARA)” considering the remarkable genotoxiccarcinogenic potential of this toxin [50]. The report of ourfindings corroborates the report of Oyedele et. al., 2017, whoalso reported a high PDI value of 91.2 ngkg−1bwday−1 forgroundnut consumers in the humid forest zones of Nigeria.A recent survey of the occurrence of AFB1 in Malaysiangroundnuts also revealed thatMalaysians are exposed to highconcentrations (24.3–34.0 ngkg−1bwday−1) of AFB1 due tohigh daily consumption rate of groundnuts in their diets[51]. Kooprasertying et al., 2016 [52], also reported highaflatoxin concentrations of peanuts sold in Thailand but atlower exposure risks (0.36–1.91 ngg−1). However, the rateof exposure to aflatoxins through consumption of aflatoxincontaminated nuts in developed countries is minimal whencompared to the situation in developing and underdevelopedcountries like Nigeria.

Liver cancer is ranked as the second and third commoncancer inmen andwomen, respectively, and the leading causeof deaths in men and the third leading cause of cancer deathsin Africa women [53]. In 2012, 12,047 cases of liver cancerwere estimated for Nigeria with age standardized incidencerates (ASR) of 11.5 per 100,000 for all causes of cancer [54].Thus it can be estimated from our result that eating aflatoxincontaminated foods could be responsible for 12% of all cancercases in Nigeria (1.38/11.5 × 100).

It was reported that the maximum tolerable limit foraflatoxins permitted in foods for liver cancer risk to beincreased by 1 out of 100,000 and 10,000 population inNigeria in a lifetime is 2 ppb and 24 ppb, respectively [11].Theconcentrations of aflatoxins in our groundnuts were higherthan the 2 ppb; thus a large percentage of the consumers areat a risk of hepatocellular carcinoma. In addition, the MOEvalues which were very low for the groundnut consumers inOgun state indicated a public health threat to the populace.Our findings also showed that consumers of cashew nuts inLagos State are at low risk of primary liver cancer

Several authors had reported the existence of a positivecorrelation between aflatoxin exposure and HCC in HBVpositive individuals [16, 35, 55]. The global burden of HCCinduced by aflatoxins was estimated to be 4.6–28.2% [16]and most cases occur in sub-Saharan Africa, Southeast Asia,and China, where populations suffer from both high HBVprevalence and largely uncontrolled exposure to aflatoxinsin foods. An assessment carried out by the Joint FAO/WHOExpert Committee on Food Additives (JECFA) to estimatethe impact of aflatoxins exposure onHCC incidence revealedthat if the hypothetical total aflatoxins permissible standardof 20𝜇gkg−1 was reduced to a stricter standard of 10 𝜇gkg−1,HCC incidence would decrease by about 300 cases per billionpeople per year in countries with an HBV prevalence of 25%[56, 57].

The inverse correlation between toxigenic isolates andaflatoxin concentrations confirms the fact that certainrequirements (suitable temperature, water activity, relative

10 Journal of Toxicology

humidity, and storage environment) must be met before theelaboration of aflatoxins in the food substances [40].

5. Conclusion

This study showed that the nuts were contaminated withpathogenic bacteria and toxigenic fungi, resulting in aflatoxincontamination of the products, thereby posing health risks totheir consumers. There is need for improvement of sanitarypractices, regular monitoring, and enforcement of standardsfor foods sold in open markets in order to reduce themenace of food borne diseases andmycotoxicoses among thepopulace. In addition, proper packaging materials and goodstorage practices should be enforced.

Conflicts of Interest

The authors disclose no conflicts of interest.

Acknowledgments

The authors are grateful to the technical staff of the Depart-ment of Biological Sciences, McPherson University, Nigeria,to the Department of Animal Health, Northwest University,Mafikeng Campus, South Africa, for their technical support,and also to National Research Foundation South Africa forthe financial support towards this research work.

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